The mechanism and control of lipoamide dehydrogenase and two related pyridine nucleotide-disulfide oxidoreductases, glutathione reductase and thioredoxin reductase are being studied. The reactions catalyzed by each of these flavoenzymes involve the transfer of two electrons via the FAD and the disulfide of a cystine residue. Peptides containing the active site cystines have been sequenced and they show structural similarities (tight loops) analogous to the mechanistic similarities in this group. A high degree of homology exists in this region of the protein between lipoamide dehydrogenase and glutathione reductase but these show no homology to thioredoxin reductase. The effects of structural modification on the activity, on the flavin chromophore, on the substrate binding sites, and on the conformational integrity of the proteins are being observed in order to further define the mechanism of action through identification of other groups crucial for function and structure. Two-electron reduction of the active center cystine of pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in their reactivity toward iodoacetamide; the reactive cysteine residue is the one nearest the amino terminus. The monoalkylated derivative is catalytically inactive toward reduced or oxidized lipoamide but is some 2-fold activated as a transhydrogenase. Extensions of this work should make assignment of specific roles to the sulfur moieties possible. The oxidation-reduction potential for the couple oxidized lipoamide dehydrogenase electron reduced enzyme has been determined as -280 mv at pH 7.6 shows that 2 protons are involved. Given that the charge transfer in 2-electron reduced enzyme is between thiolate and FAD, this indicates that a base is essential at the active site to deprotonate the dithiol substrate and then to stabilize the charge transfer thiolate. BIBLIOGRAPHIC REFERENCES: Active Center Sequence Homology and Mechanistic Homology Between Glutathione Reductase and Lipoamide Dehydrogenase, C.H. Williams, Jr., L.D. Arscott and E.T. Jones, Flavins and Flavoproteins, T.P. Singer, ed., Elsevier Scientific Publishing Co., Amsterdam, 1976, pp. 455-463.